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Effect of the NF-κB/iNOS Signaling Pathway on Spiral Ganglion in Mouse Model of Sensorineural Hearing Loss
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摘要:目的 探究在3-硝基丙酸（3-NP）诱导的感音神经性听力损失小鼠模型中核转录因子κB（NF-κB）/诱导型一氧化氮合酶（iNOS）信号通路的表达水平变化对听力损失的影响。方法 本研究采用鼓室注射法进行动物建模。将C57BL/6雄性小鼠分为3个组，每组9只。3-NP组（鼓室内注射3-NP溶液），3-NP+EVP4593组（鼓室内注射3-NP溶液，腹腔注射NF-κB抑制剂EVP4593溶液），以及对照组〔鼓室内注射磷酸缓冲液（PBS）〕。在注射前后进行听性脑干反应检测。4周后取出耳蜗，并进行NF-κB p65、RelB、iNOS、Caspase-3抗体的免疫组织化学及实时荧光染色定量（qRT）-PCR检测。结果 3-NP组小鼠反应阈值较对照组和3-NP+EVP4593组高（P<0.05），且3-NP+EVP4593组的听阈比对照组高（P<0.05）。免疫荧光染色和qRT-PCR结果显示3-NP暴露可导致螺旋神经节区域NF-κB p65和RelB、iNOS表达较对照组增加（P<0.05），其表达可随着EVP4593的应用而减少（P<0.05）。3-NP组螺旋神经节细胞Caspase-3表达高于对照组，3-NP+EVP4593组Caspase-3的表达较3-NP组减少（P<0.05）。结论 3-NP可通过激活NF-κB/iNOS信号通路导致感音神经性听力损失模型小鼠的耳蜗螺旋神经节发生炎症反应，这可能在感音神经性听力损失的发病中起重要作用。Abstract:Objective To explore the effect of changes in the expression level of necorsis factor (NF)-κB/inducible nitric oxide synthase (iNOS) signaling pathway on hearing loss in a mouse model of sensorineural hearing loss (SNHL) induced by 3-nitropropionic acid (3-NP).Methods The animal model was established by tympanic injection. C57BL/6 male mice were divided into three groups, 3-NP group receiving tympanic injection of 3-NP solution, 3-NP+EVP4593 group receiving tympanic injection of 3-NP solution and intraperitoneal injection of EVP4593 solution, and a control group receiving tympanic injection of phosphate buffered saline (PBS). Auditory brainstem response (ABR) was tested before and after injection. After 4 weeks, the cochlea was harvested and immunohistochemistry and qRT-PCR of NF-κB p65, RelB, iNOS, and Caspase-3 were conducted accordingly.Results The hearing thresholds of the 3-NP group were higher than those of the control group and the 3-NP+EVP4593 group (P<0.05), and the hearing thresholds of the 3-NP+EVP4593 group were also higher than those of the control group (P<0.05). Immunofluorescence staining and qRT-PCR results showed that 3-NP exposure caused an increase in the expressions of NF-κB p65, RelB, and iNOS in the spiral ganglion in comparison with those of the control group (P<0.05), and their expressions decreased with the administration of EVP4593 (P<0.05). The expression of Caspase-3 in the spiral ganglion cells in the 3-NP group was higher than that in the control group, while in the 3-NP+EVP4593 group, it was lower than that in the 3-NP group (P<0.05).Conclusion This study found that, by activating the NF-κB/iNOS signaling pathway, 3-NP may cause inflammation in the spiral ganglion of the cochlear in the SNHL model mice, which may play an important role in the pathogenesis of SNHL.
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Keywords:
- 3-nitropropionic acid /
- Sensorineural hearing loss /
- Spiral ganglion /
- NF-κB /
- iNOS
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图 1 小鼠ABR听力反应阈值变化
Figure 1. ABR threshold shifts after exposure to 3-NP and 3-NP plus EVP4593
* P<0.05, vs. control group; Δ P<0.05, vs. 3-NP+EVP4593 group. n=3.
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图 2 耳蜗螺旋神经节NF-κB p65、RelB、iNOS及Caspase-3的表达
Figure 2. The expression of NF-κB p65, RelB, iNOS, and Caspase-3 in the spiral ganglion
White arrow: Nuclear translocation of NF-κB p65.
表 1 qRT-PCR引物序列
Table 1 Primer sequences for qRT-PCR
Target gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′) Production length/bp NF-κB p65 CTTCCTCAGCCATGGTACCTCT CAAGTCTTCATCAGCATCAAACTG 126 RelB CTTTGCCTATGATCCTTCTGC GAGTCCAGTGATAGGGGCTCT 122 iNOS CTATCAGGAAGAAATGCAGGAGAT GAGCACGCTGAGTACCTCATT 125 GAPDH GGTGAAGGTCGGTGTGAACG CTCGCTCCTGGAAGAATGGTG 121 
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表 2 耳蜗螺旋神经节iNOS、NF-κB 65和RelB的表达平均荧光强度值及基因表达变化
Table 2 The mean fluorescence intensity and expression trend of iNOS, NF-κB p65, and RelB in the spiral ganglion
Index Group n Mean fluorescence intensity (protein) n Relative expression (gene) iNOS Control 6 62.04±1.81# 3 1.05±0.07# 3-NP 6 82.99±2.61 3 1.80±0.04 3-NP+EVP4593 6 58.87±1.98# 3 1.15±0.06# NF-κB p65 Control 6 70.03±2.94# 3 0.95±0.09# 3-NP 6 95.27±2.15 3 1.85±0.04 3-NP+EVP4593 6 68.21±3.52# 3 1.24±0.17#, Δ RelB Control 6 35.50±3.95# 3 1.10±0.03# 3-NP 6 104.40±4.66 3 2.20±0.10 3-NP+EVP4593 6 66.00±3.88#, Δ 3 1.20±0.16# # P<0.05, vs. 3-NP group; Δ P<0.05, vs. control group.
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